Journal: Human Molecular Genetics

Article Title: Interleukin-10 reduces the pathology of mdx muscular dystrophy by deactivating M1 macrophages and modulating macrophage phenotype

doi: 10.1093/hmg/ddq523

Figure Lengend Snippet: IL-10 modulates muscle cell differentiation in vitro and in vivo. (A) Myoblasts were cultured without addition of exogenous factors (‘none') or in the presence of IL-10, LPS, LPS and IL-10 together or bFGF, and then assayed for cell number at the end of 3 days of stimulation. Neither IL-10, LPS or IL-10 combined with LPS affected proliferation, although bFGF, which was used as a positive control, increased cell number by nearly 50% under the same culture conditions. (B) Pre-treatment of macrophages with IFN-γ and TNF-α prior to co-culture with myoblasts decreased myoblast numbers at the end of a 3-day period of co-culture, compared with myoblasts cultured alone (‘none'). Pre-treatment of macrophages with IL-10 or IL-4 combined with IL-13 to induce M2c phenotype generated macrophages that promoted myoblast proliferation in co-cultures. n = 6 for each treatment group. Asterisks indicate significant difference from the ‘none' group at P< 0.05. Bars represent sem. (C) Quantitative, real-time PCR results for relative mRNA levels for Pax7 in quadriceps muscles of 4-week-old or 12-week-old mdx mice that expressed IL-10 (+/+) or were IL-10 null mutants (−/−). Expression levels are relative to 4-week-old IL-10+/+/mdx muscles, for which the expression level is set at one unit. Each experimental group included quadriceps from five mice. Bars represent sem. (D) Western blots of extracts of myoblast cultures collected 0–7 days after initiating cultures in the presence of IL-10 or addition of no exogenous cytokine (‘none'). Blots were probed with antibodies to MyoD or myogenin. The membrane used for immunoblotting was stained with Ponceau red (‘Loading') before application of the antibody to verify equal protein loading in each lane. (E and F) Quantitative, real-time PCR results for relative mRNA levels for MyoD (E) or myogenin (F) in quadriceps muscles of 4-week-old or 12-week-old mdx mice that expressed IL-10 (+/+) or were IL-10 null mutants (−/−). Expression levels are relative to 4-week-old IL-10+/+/mdx muscles, for which the expression level is set at one unit. Each experimental group included quadriceps from five mice. Hash symbol indicates significant difference from age-matched IL-10+/+/mdx muscles. Asterisks indicate significant difference from 4-week-old IL-10+/+/mdx muscles. Bars represent sem.

Article Snippet: Peritoneal macrophages were seeded at 4.0×10 5 cells/well in six-well plates and stimulated with mouse recombinant IFN-γ (10 ng/ml, BD Pharmingen) and TNF-α (10 ng/ml, BD Pharmingen) or IL-10 (10 ng/ml, BD Pharmingen) or IL-4 (10 ng/ml, BD Pharmingen) and IL-13 (10 ng/ml, BD Pharmingen) in DMEM containing 0.25% FBS and 1% penicillin–streptomycin (P/S) for 24 h at 37°C and 5% CO 2 .

Techniques: Cell Differentiation, In Vitro, In Vivo, Cell Culture, Positive Control, Co-Culture Assay, Generated, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Staining

Journal: Human Molecular Genetics

Article Title: Interleukin-10 reduces the pathology of mdx muscular dystrophy by deactivating M1 macrophages and modulating macrophage phenotype

doi: 10.1093/hmg/ddq523

Figure Lengend Snippet: IL-10 induces phagocytosis by macrophages and clodronate depletion of phagocytes from mdx mice selectively reduces M2c macrophages in skeletal muscle. (A–D) Flow cytometric data showing that IL-10 stimulation of macrophages increases macrophage phagocytosis. Macrophages were untreated with exogenous cytokines (A), treated with IFN-γ (B), IL-4 (C) or IL-10 (D) prior to incubation with flourescent microspheres for phagocytosis. Cells were then analyzed by flow cytometry to determine the proportion of cells that contained fluorescent microspheres, as an index of phagocytosis. Neither IFN-γ (68.1%) or IL-4 (61.2%) increased the proportion of cells that were phagocytic, compared with untreated controls (69.4%). However, IL-10 treatments increased the proportion of cells that were phagocytic (79.5%). (E and F) Intraperitoneal injections of clodronate-containing liposomes significantly reduced the numbers of macrophages (E) and MHC-2-presenting cells in the quadriceps muscles of 4-week-old mdx mice. Each experimental group included muscles from six mice. Asterisks indicate a significant difference from PBS-treated controls. Bars represent sem. (G–J) Quantitative, real-time PCR data show that clodronate-mediated depletions of phagocytes did not cause a significant change in the expression of CD68 in the quadriceps muscles of 4-week-old mdx mice (G), but caused large, significant reductions in the expression of CD206 (H), CD163 (I) and IL-10 (J). Each experimental group included muscles from five mice. Asterisks indicate a significant difference from PBS-treated controls. ‘n.s.' indicates no significant effect of the clodronate treatment. Bars represent sem, which is too small to appear for graphs of some data sets.

Article Snippet: Peritoneal macrophages were seeded at 4.0×10 5 cells/well in six-well plates and stimulated with mouse recombinant IFN-γ (10 ng/ml, BD Pharmingen) and TNF-α (10 ng/ml, BD Pharmingen) or IL-10 (10 ng/ml, BD Pharmingen) or IL-4 (10 ng/ml, BD Pharmingen) and IL-13 (10 ng/ml, BD Pharmingen) in DMEM containing 0.25% FBS and 1% penicillin–streptomycin (P/S) for 24 h at 37°C and 5% CO 2 .

Techniques: Incubation, Flow Cytometry, Real-time Polymerase Chain Reaction, Expressing